In vitro action of sho-seiryu-to on allergen-exposed mononuclear cells.

Although Sho-seiryu-to (SST), used as a traditional herbal (Kampo) medicine mainly in China and Korea, is shown to have immunomodulating potential, such as an anti-allergic one, its underlying mechanism has not been completely clarified. To partially address the issue, we explored its effects on allergen-exposed mononuclear cells. Male balb/c mice were intraperitoneally administered ovalbumin (OVA: 20 μg) plus alum or vehicle twice (Day 0 and Day 14). At Day 21, mice were sacrificed and splenocytes (mononuclear cells) were isolated and cultured in the presence or absence of OVA with or without SST. Thereafter, helper T-related cytokines in the culture supernatants were evaluated by means of ELISA. Protein level of interferon-γ was lower than 5.0 pg/mL in the supernatants from OVA– non-exposed or -exposed mononuclear cells in the presence or absence of OVA stimulation. On the other hand, SST induced the cytokine from both types of mononuclear cells in the presence (P < 0.05) or absence of OVA stimulation as compared to corresponding control. By contrast, interleukin (IL)-4 level tended to be decreased by SST in OVA-non-exposed mononuclear cells as did IL-13 in both non-exposed and exposed mononuclear cells as compared to vehicle. In conclusion, immunoregulating efficacy by SST on allergy-prone subjects may include, at least in part, restoring helper T balance mainly through hyperproduction of IFN-γ against mononuclear cells such as lymphocytes.

Variations in the structure and transcription of the mitochondrial atp and cox genes in wild Solanum species that induce male sterility in eggplant (S. melongena).

In order to determine the molecular basis of cytoplasmic male sterility (CMS) in alloplasmic lines of eggplant, the genomic structures and transcription patterns of mitochondrial ATP synthase subunit (atp) and cytochrome oxidase subunit (cox) genes were studied for wild and cultivated eggplants. Alloplasmic eggplant lines with cytoplasms of wild Solanum species showing either anther indehiscent type of CMS or non-pollen production type of CMS were studied with the cultivated eggplant Solanum melongena, used as a control. Southern hybridization of the mitochondrial genes indicated the difference between the two types of CMS and showed complete identity within each type. The cytoplasmic patterns of all wild species differed from that of the cultivated eggplant. Thus, the cytoplasm of the six wild eggplants and the one cultivated eggplant was classified into three groups. Male sterile plants of both types of CMS showed novel transcription patterns of atp1, whereas a different transcription pattern of cox2 was observed only in the anther indehiscent type. Based on these differences, we determined the DNA sequences of about a 4 kbp segment in the atp1 region. Although the coding and 3' flanking regions were almost identical among the cytoplasms, the 5' flanking region was completely different and novel open reading frames (orfs) were found for each of the CMS types and the cultivated eggplant. The cytoplasm of Solanum kurzii inducing the anther indehiscent type CMS had orf312, and those of Solanum aethiopicum and Solanum grandifolium of non-pollen production type CMS had orf218. The correspondence between the transcription patterns of these orfs and phenotypic expression of male sterility strongly suggests that these orfs are causal genes for each type of CMS.

Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice.

AIMS/HYPOTHESIS: The pancreas and hypothalamus are critical for maintaining nutrient and energy homeostasis, and combined disorders in these organs account for the onset of the metabolic syndrome. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. The physiological role of ATF3 in the pancreas has been controversial, and its role in the hypothalamus remains unknown. To elucidate the roles of ATF3 in these organs, we generated pancreas- and hypothalamus-specific Atf3 knockout (PHT-Atf3-KO) mice in this study. METHODS: We crossed mice bearing floxed Atf3 alleles with Pdx1-cre mice, in which cre is specifically expressed in the pancreas and hypothalamus, and analysed metabolic variables, pancreatic morphology, food intake, energy expenditure and sympathetic activity in adipose tissue. We also used a hypothalamic cell line to investigate the molecular mechanism by which ATF3 regulates transcription of the gene encoding agouti-related protein (Agrp). RESULTS: Although PHT-Atf3-KO mice displayed better glucose tolerance, neither plasma glucagon nor insulin level was altered in these mice. However, these mice exhibited higher insulin sensitivity, which was accompanied by a leaner phenotype due to decreased food intake and increased energy expenditure. We also observed decreased hypothalamic Agrp expression in PHT-Atf3-KO mice. Importantly, an increase in ATF3 levels is induced by fasting or low glucose in the hypothalamus. We also showed that ATF3 interacts with forkhead box-containing protein, O subfamily 1 (FoxO1) on the Agrp promoter and activates Agrp transcription. CONCLUSIONS/INTERPRETATION: Our results suggest that ATF3 plays an important role in the control of glucose and energy metabolism by regulating Agrp.

Analysis of motor function modulated by cholinergic neurons in planarian Dugesia japonica.

Recent studies of the freshwater planarian Dugesia japonica have revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. Here, we identified the gene for planarian choline acetyltransferase (Djchat), which is essential for acetylcholine (ACh) biosynthesis. Immunofluorescence studies using anti-Dugesia japonica ChAT (DjChAT) antibody revealed that cholinergic neurons are widely distributed in the planarian nervous system, including the brain, ventral nerve cords, optic nerves, and pharyngeal nerve plexus. In order to investigate the function of cholinergic neurons in planarians, we used both pharmacological and RNA interference (RNAi) approaches. Administration of physostigmine (an acetylcholinesterase inhibitor) clearly elevated the amount of ACh, and then induced sudden muscle contraction behavior in a concentration-dependent manner. In addition, we found that pretreatment with tubocurarine (a muscle nicotinic ACh receptor antagonist) or atropine (a non-selective muscarinic ACh receptor antagonist), but not pretreatment with mecamylamine (a neural nicotinic ACh receptor antagonist), significantly extended the latency time for physostigmine-induced contraction behavior, suggesting that muscle nicotinic ACh receptors and muscarinic ACh receptors contribute to physostigmine-induced contraction behavior. We also confirmed that ACh biosynthesis ability and DjChAT-immunoreactivity were eliminated in Djchat(RNAi) planarians. Moreover, the decrease of the level of ACh induced by Djchat(RNAi) caused extension of the latency time for contraction behavior. Our findings support the possibility that the cholinergic functions of planarians are similar to those of vertebrates, suggesting that planarians are simple but useful model organisms for getting insight into the cholinergic nervous system in higher animals.

Preseasonal prophylactic treatment with antihistamines suppresses nasal symptoms and expression of histamine H1 receptor mRNA in the nasal mucosa of patients with pollinosis.

Administration of antihistamines 2-4 weeks before the pollen season showed a greater inhibitory effect on nasal allergy symptoms in patients with seasonal allergic rhinitis. However, the mechanism of slow-onset effects of preseasonal treatment with antihistamines remains unclear. Here, we investigated the effect of preseasonal prophylactic treatment with antihistamines on nasal symptoms and the expression of histamine H1 receptor (H1R) mRNA of the nasal mucosa in patients with cedar pollen pollinosis. During the peak pollen period, the expression of H1R mRNA in the nasal mucosa and the scores of sneezing and watery rhinorrhea in patients receiving preseasonal prophylactic treatment with antihistamines were significantly suppressed in comparison with those in the patients without treatment. Moreover, there was a significant correlation between the nasal symptoms and the expression of H1R mRNA in both patients with or without preseasonal prophylactic treatment. These findings suggest that preseasonal prophylactic treatment with antihistamines is more effective than on-seasonal administration to patients with pollinosis in reducing nasal symptoms during the peak pollen period by suppressing H1R gene expression in the nasal mucosa.

A subchronic toxicity study of dunaliella carotene in F344 rats.

Dunaliella carotene, extracted from dunaliella alga (Dunaliella bardawil or Dunaliella salina), for use as a food-coloring agent, has beta-carotene as its mainly constituent. As there have been no reports of toxicological evaluation, a 90-day subchronic toxicity study was here performed in F344 rats at dose levels of 0 (control), 0.63%, 1.25%, 2.5% and 5% in powdered basal diet. The average daily intakes of dunaliella carotene were 352, 696, 1420 and 2750 mg/kg/day, respectively, for males, and 370, 748, 1444 and 2879 mg/kg/day for females. No mortality or treatment-related clinical signs were observed throughout the experimental period in any of the groups. Body weight gain was slightly but significantly (p < 0.05) reduced from week 5 to the end of the experiment in 2.5% and 5% males. Increased PLT were observed in 1.25% and 5% males, and 2.5% and 5% females. Significant elevations or tendencies for increase in serum T. Cho and Ca were observed in all treated males and females, with clear dose-dependence in males. Organ weight measurement and histopathological observation revealed no toxicological changes. Based on growth suppression, no-observed-adverse-effect-levels (NOAELs) were estimated to be 1.25% (696 mg/kg/day) for males and 5% (2879 mg/kg/day) for females. As increases in serum Ca were observed in the lowest group in both sexes, a no-observed-effect level (NOEL) could not be determined in this study.

Lack of chemopreventive effects of alpha-eleostearic acid on 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis in female Sprague-Dawley rats.

alpha-Eleostearic acid is one of the conjugated linolenic acids from tung oil, which is obtained from the seeds of Aleurites fordii. The effects of dietary alpha-eleostearic acid (18:3, n-5) on the post-initiation period of 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis were examined using female Sprague-Dawley (SD) rats. For initiation, rats were given subcutaneous injections of 40mg/kg body weight (5 times) and 20mg/kg body weight (3 times) of DMH during the age of 6-8 weeks and a single intragastric administration of 50mg/kg body weight of DMBA at 9 weeks. Then, the animals were treated with 0%, 0.01%, 0.1% or 1.0% alpha-eleostearic acid for 34 weeks. Control rats received the basal diet alone or 1.0% alpha-eleostearic acid without prior initiation treatment. All surviving animals were killed at week 37 of the experiment. There were no statistically significant alterations in any of the parameters for either mammary or colon tumors. These results thus indicate that alpha-eleostearic acid does not exert clear modification effects on DMBA and DMH-induced mammary and colon carcinogenesis, at least under the present experimental conditions.

Lack of subchronic toxicity of an aqueous extract of Agaricus blazei Murrill in F344 rats.

Agaricus blazei Murrill, an edible mushroom, is widely used as a functional food due to its possible medicinal effects. Aqueous extracts are also used as food additive to provide an agreeable bitter taste. As a part of its safety assessment, the present 90-day subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), rats were fed powder diet containing A. blazei Murrill aqueous extract at dose levels of 0 (basal diet), 0.63, 1.25, 2.5 and 5% (maximum) for 90 days. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum blood urea nitrogen was slightly but significantly increased in males of the 2.5 and 5% groups, no related histopathological changes were observed in the kidney, and serum creatinine levels were rather reduced, suggesting the increase of blood urea nitrogen to be of little toxicological significance. Hematology, organ weight measurement and histopathological observation revealed no test compound-related toxicological changes. In conclusion, A. blazei Murrill extract even at 5% in the diet (2654 mg/kgb.w./day for male rats and 2965 mg/kgb.w./day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL was concluded to be 5% in the diet.

Penicillin derivatives induce chemical structure-dependent root development, and application for plant transformation.

We investigated five penicillin derivatives that are popularly used for transformation experiments with Agrobacterium rhizogenes-penicillin G, carbenicillin, ampicillin, amoxicillin and cephalexin-for their effects on the growth and morphology of Beta vulgaris, Capsicum annuum and Glehnia littoralis roots. Attention was given to the relationship between their chemical structures and functions. Ampicillin was found to stimulate root elongation but inhibit root branching, whereas carbenicillin inhibited root elongation but promoted root branching. Root cultures were also exposed to hydrolyzed products of these antibiotics-i.e. phenylmalonic acid (PM), phenylglycine and 6-aminopenicillanic acid (6-APA): PM inhibited root elongation the most, while root elongation was supported best by 6-APA. These results indicate that both the side chains and the major component of penicillin derivatives affect root development and that the nature of the side chains is responsible for the responses. Ampicillin but not carbenicillin was used in subsequent experiments described herein to eliminate bacteria and to support root growth of transformants of the recalcitrant plants.

A subchronic toxicity study of shea nut color in Wistar rats.

Shea nut color, obtained from nuts of the shea tree (Butyrospermum parkii), is used as a food-coloring agent. Flavonoid pigments are considered to be the responsible constituents. As there have been no reports of toxicological evaluation, a 13-week subchronic toxicity study was performed in Wistar Hannover rats at dose levels of 0 (control), 0.07, 0.31, 1.25 and 5% in powdered basal diet. The average of daily shea nut color intake was 51.3, 226.1, 986.8 and 3775.5 mg/kg/day for males and 56.4, 272.9, 1166.7 and 4387.7 mg/kg/day for females, respectively. During the administration period, daily observation of clinical signs and weekly measurement of body weights and food consumption were performed. After the end of the treatment, hematology, serum biochemistry, organ weight and histopathological examinations were conducted. No significant toxicological changes were observed in any parameters in this study. Hence, the no adverse effect dose of shea nut color was estimated to be greater than 5.0% for both sexes (3775.5 mg/kg/day for males and 4387.7 mg/kg/day for females).

Lipopolysaccharide-induced switch between retinoid receptor (RXR) alpha and glucocorticoid attenuated response gene (GARG)-16 messenger RNAs in cultured rat microglia.

Glucocorticoid-attenuated response genes (GARG) belong to a recently described family of genes responsive to the action of dexamethasone. Full-length cDNA of one member of this family, GARG16, has been cloned from rat microglia and regulation of its mRNA expression has been studied. Moreover, regulation of retinoid/retinoic acid activated transcription factor (RXR/RAR) mRNAs in mixed astrocyte and in purified microglia cultures has been investigated. RARbeta mRNA was undetectable in microglia by RT-PCR, whereas clearly present in the mixed cultures. RXRalpha, RARgamma, and GARG16 mRNAs were found in both culture systems. RXRalpha mRNA was strongly expressed in control microglia but rapidly declined upon treatment with LPS. Conversely, GARG16 mRNA was almost untraceable in control microglia but rapidly increased by LPS. Time-course studies revealed an oscillating behavior of expression of both mRNAs during the first 6 hr, which receded to control levels (RXRalpha high, GARG16 low) at 72 hr of LPS-treatment. Additionally, p38 MAPK and SEK phosphorylations peaked at 1 hr followed by steady declines, whereas MEK and c-Jun showed double peaks at 1+4 hr and 1+6 hr, respectively, before subsiding to control levels. This behavior was not observed in comparative studies with TNF-alpha, interleukin-10 (IL-10), or interferon-gamma inducible protein 10 (IP-10). Finally, inhibitors of p38 MAPK, p42/p44 ERK, and PKCalpha as well as the use of dexamethasone revealed major influences of the p38 MAPK-c-Jun-AP-1 signaling pathway on RXRalpha and GARG16 mRNA expressions. The counter regulatory control of GARG16 and RXRalpha mRNA expression is believed to be an example of a fine-tuned cellular mechanism to react to inflammatory stimuli.

The binding site of transcription factor YY1 is required for intramolecular recombination between terminally repeated sequences of linear replicative hepatitis B virus DNA.

In the replication cycle of hepadnavirus DNA, the double-stranded linear form of viral DNA is generated as a minor replicative intermediate, which is efficiently converted to covalently closed circular DNA (cccDNA) by intramolecular recombination (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). We previously found a binding site of transcription factor Yin and Yang 1 (YY1) in one terminal region of the double-stranded linear replicative hepatitis B virus (HBV) DNA (M. Nakanishi-Matsui, Y. Hayashi, Y. Kitamura, and K. Koike, J. Virol. 74:5562-5568, 2000). However, it is not known whether the YY1-binding site is required for the intramolecular recombination of HBV DNA. In this study, we established an HBV-producing system in which the cccDNA appeared to be generated from the transfected linear DNA or the linear replicative DNA by nonhomologous end joining (NHEJ) or by both NHEJ and homologous recombination between terminally repeated sequences, respectively. When the YY1-binding site in the terminal region of transfected linear viral DNA was mutated, the cccDNA was generated merely by NHEJ. Results suggest that the YY1-binding site in the terminal region of linear replicative HBV DNA is required for intramolecular recombination between terminally repeated sequences.

Integrated hepatitis B virus DNA preserves the binding sequence of transcription factor Yin and Yang 1 at the virus-cell junction.

Accumulated findings have indicated that hepatitis B virus (HBV) DNA integrates into the cellular DNA of HBV-infected chronic hepatitis tissues. The integrated sequence (IS) of HBV DNA at the virus-cell junction is conserved in a 25-bp region which is adjacent to direct repeat 1. A cellular protein which we purified from the nuclear extract of HepG2 cells binds to the IS and was designated IS binding protein 3 (ISBP3). The amino acid sequence of ISBP3 was determined and found to be identical to that of transcription initiation factor Yin and Yang 1 (YY1). An antibody against C-terminal amino acids of YY1 recognized ISBP3 in a Western blot analysis and an electrophoretic mobility shift assay. Furthermore, ISBP3 also interacted with Y3, which corresponds to the YY1 binding sequence, to enhance intramolecular recombination of polyomavirus DNA. Although YY1 is known as a transcription factor, the IS did not exhibit any effect on the transcription of precore and pregenome RNAs. The possible involvement of YY1 in the intramolecular recombination of linear replicative HBV DNA has been examined (Y. Hayashi et al., unpublished data). Data suggest that YY1 is involved in the joining reaction between HBV DNA and cellular DNA to form the virus-cell junction.

Cbfa1 is a positive regulatory factor in chondrocyte maturation.

Cbfa1 is a transcription factor that belongs to the runt domain gene family. Cbfa1-deficient mice showed a complete lack of bone formation due to the maturational arrest of osteoblasts, demonstrating that Cbfa1 is an essential factor for osteoblast differentiation. Further, chondrocyte maturation was severely disturbed in Cbfa1-deficient mice. In this study, we examined the possibility that Cbfa1 is also involved in the regulation of chondrocyte differentiation. mRNAs for both Cbfa1 isotypes, type I Cbfa1 (Pebp2alphaA/Cbfa1) and type II Cbfa1 (Osf2/Cbfa1 or til-1), which are different in N-terminal domain, were expressed in terminal hypertrophic chondrocytes as well as osteoblasts. In addition, mRNA for type I Cbfa1 was expressed in other hypertrophic chondrocytes and prehypertrophic chondropcytes. In a chondrogenic cell line, ATDC5, the expression of type I Cbfa1 was elevated prior to differentiation to the hypertrophic phenotype, which is characterized by type X collagen expression. Treatment with antisense oligonucleotides for type I Cbfa1 severely reduced type X collagen expression in ATDC5 cells. Retrovirally forced expression of either type I or type II Cbfa1 in chick immature chondrocytes induced type X collagen and MMP13 expression, alkaline phosphatase activity, and extensive cartilage-matrix mineralization. These results indicate that Cbfa1 is an important regulatory factor in chondrocyte maturation.

Inhibitory effect of mast cell-mediated immediate-type allergic reactions by Cichorium intybus.

We investigated the effect of an aqueous extract of Cichorium intybus (CIAE) on mast cell-mediated immediate type allergic reactions. CIAE (0.1-1000 mg kg-1) dose-dependently inhibited systemic anaphylactic reaction induced by compound 48/80 in mice. Especially, CIAE inhibited compound 48/80-induced anaphylactic reaction 100% with the dose of 1000 mg kg-1. CIAE 1000 mg kg-1also significantly inhibited local anaphylactic reaction activated by anti-dinitrophenyl (DNP) IgE. When mice were pretreated with CIAE at a concentration ranging from 0.1 to 1000 mg kg-1, the plasma histamine levels were reduced in a dose-dependent manner. CIAE (1-1000 microg ml-1) dose-dependently inhibited histamine release from the rat peritoneal mast cells (RPMC) activated by compound 48/80 or anti-DNP IgE. The level of cAMP in RPMC, when CIAE (1000 microg ml-1) was added, increased significantly compared with that of control cells. These results indicate that CIAE inhibits mast cell-mediated immediate-type allergic reactions in vivo and in vitro.

Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection.

In this study, we analyzed the mechanism of selective motor neuronal death, a characteristic of amyotrophic lateral sclerosis, using embryonic rat spinal cord culture. When dissociated cultures were exposed to low-level glutamate (Glu) coadministered with the Glu transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC) for 24 hours, motor neurons were selectively injured through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. Nitric oxide synthase (NOS) inhibitors attenuated this toxicity, and long-acting nitric oxide (NO) donors damaged motor neurons selectively. Nonmotor neurons survived after exposure to low-dose Glu/PDC, but Glu-induced toxicity was potentiated by coadministration of an NO-dependent guanylyl cyclase inhibitor. In addition, 8-bromo-cyclic GMP, a soluble cyclic GMP analogue, rescued nonmotor neurons, but not motor neurons, exposed to high-dose Glu/PDC. Twenty-four hours' incubation with PDC elevated the number of neuronal NOS-immunoreactive neurons by about twofold compared with controls, and a double-staining study, using the motor neuron marker SMI32, revealed that most of them were nonmotor neurons. These findings suggest that selective motor neuronal death caused by chronic low-level exposure to Glu is mediated by the formation of NO in nonmotor neurons, which inversely protects nonmotor neurons through the guanylyl cyclase-cyclic GMP cascade. Induction of neuronal NOS in nonmotor neurons might enhance both the toxicity of motor neurons and the protection of nonmotor neurons, which could explain the pathology of amyotrophic lateral sclerosis.

Inhibition of immediate type allergic reactions by the aqueous extract of Kum-Hwang-San.

We studied the effect of aqueous extract of Kum-Hwang-San (KHS) on mast cell-mediated immediate type allergic reactions. KHS (1-100 microg/site) inhibited concentration-dependently mast cell-dependent ear swelling response induced by compound 48/80 (200 microg/site) in mice by both topical and intradermal application. KHS (0.1-100 microg/site) inhibited concentration-dependently passive cutaneous anaphylaxis induced by anti-dinitrophenyl (DNP) IgE in rats by both topical and intradermal application. KHS also inhibited concentration-dependently the histamine release from the rat peritoneal mast cells (RPMC) by compound 48/80 and anti-DNP IgE. Moreover, KHS had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha (TNF-alpha) secretion from RPMC. These results indicate that KHS inhibits immediate type allergic reactions by inhibition of histamine release and TNF-alpha secretion from mast cells in vivo and in vitro.

Induction of furanocoumarin biosynthesis in Glehnia littoralis cell suspension cultures by elicitor treatment.

Cell suspension cultures were established from Glehnia littoralis plants belonging to two different geographic strains. When the cells were treated with yeast extract, they started to produce and excrete furanocoumarins into the culture medium; a major component, bergapten, and a minor one, xanthotoxin, were detected and identified by HPLC and GC/MS. Changes in phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production after elicitor treatment were traced, showing that PAL activity increased rapidly, reached a maximum after 24 h, and then declined to the normal level after 96 h which preceded the induced bergapten production. The induced-PAL activity of the cultured cells established from an S-type plant which accumulated trace amounts of furanocoumarins was about 50% of that in the cultured cells from an N-type plant that accumulated more than 0.1% furanocoumarins in the underground parts. However, the elicited production of bergapten was about six times higher in the cell cultures from the S-type plant. Addition of the PAL inhibitor 2-aminoindan-2-phosphoric acid (AIP) at 10 microM suppressed the induction of PAL activity and furanocoumarin production.

Impaired expression of noncollagenous bone matrix protein mRNAs during fracture healing in ascorbic acid-deficient rats.

In scorbutic patients, fractures are slow to heal because of impaired collagen synthesis. To investigate the influence of impaired collagen synthesis on the differentiation and proliferation of osteogenic and chondrogenic cells, we examined the expression of genes encoding bone matrix proteins, including osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP), as differentiation markers for osteogenic and chondrogenic cells during fracture healing in Osteogenic Disorder Shionogi (ODS) rats, which have a hereditary defect in the ability to synthesize ascorbic acid (Asc). In ODS rats without Asc supplementation, intramembranous ossification was completely inhibited. Although a few fibroblast-like cells expressing ON mRNA were observed, no OPN mRNA-expressing cells were detected. During endochondral ossification, a small amount of metachromatic staining cartilage appeared at the fracture site, but there was no provisional calcification zone in the cartilage. Chondrocytes expressed ON and MGP mRNAs, but not OPN mRNA. When Asc was given to these rats, callus formation was soon detected around the fracture site, while OPN mRNA was expressed by differentiated osteoblasts and hypertrophic chondrocytes. Our data indicate that impaired collagen synthesis due to Asc deficiency inhibited the increase of ON and MGP mRNA-expressing cells as well as the appearance of OPN mRNA-expressing cells. Since OPN is considered to play an important role in normal and pathological mineralization, lack of OPN mRNA expression accompanying impaired collagen synthesis may have a role in defective mineralization and delayed fracture healing in scurvy.

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3.

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.